Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity.
The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.
Baldinger
Tina
T
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Gossen
Manfred
M
D000705:Anaphase; D000818:Animals; D030801:Animals, Genetically Modified; D002875:Chromosomes / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology* / Q000196:embryology / Q000201:enzymology*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019076:Transgenes
D029721:Drosophila Proteins; C497216:Orc2 protein, Drosophila; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018844:Cyclin-Dependent Kinases